No abstract

We evaluated the mechanism of uptake of carboxyfluorescein-containing small unilamellar liposomes of different surface charge by trophoblast cells in culture. Carboxyfluorescein-encapsulated neutral liposomes were prepared by using equimolar concentrations of lecithin and cholesterol. Anionic and cationic liposomes were prepared by adding dicetylphosphate and stearylamine. Trophoblast cells from human term placenta were cultured and incubated on the first day at 37 degrees C with liposome-encapsulated carboxyfluorescein or 500 nM of free carboxyfluorescein. The mechanism of uptake was determined by pre-treating the cells with metabolic inhibitors: 2 mM of sodium azide and 25 mM of deoxyglucose for 30 min. The uptake of liposomes was also evaluated both qualitatively under fluorescent microscope and quantitatively by measuring carboxyfluorescein fluorometrically. The uptake of free carboxyfluorescein and cationic liposomes was comparable. The anionic liposomes were taken up by the trophoblast cells more avidly than the neutral (13.2 +/- 1.6 versus 9.5 +/- 1.4%; P <0.01), cationic (2.9 +/- 0.4%; P <0.001) and the free carboxyfluorescein (2.1 +/- 0.9%; P <0.01). When cells were pre-treated with metabolic inhibitors, the uptake of anionic (5.9 +/- 1.8%; P <0.001) and neutral liposomes (4.0 +/- 0.8%; P <0.01) was significantly reduced, whereas uptake of cationic and free carboxyfluorescein remained unaltered. This study indicates that small unilamellar liposomes are internalized by the trophoblast cells in culture by an energy-dependent pathway; most probably by endocytosis. The neutral and anionic liposomes are internalized more avidly than cationic liposomes.

Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.

Selected lobules of term human placenta were extracorporeally perfused and human immunoglobulin-G complexed to horseradish peroxidase (IgG-HRP) was added to the maternal perfusate. After different durations of perfusion IgG-HRP was visualised by use of diamino-benzidine cytochemistry. Within the first 10 min of perfusion IgG-HRP was found bound to microvilli and coated pits of the syncytiotrophoblast; internalisation into coated vesicles and tubulo-vesicular bodies was also observed. Subsequently, IgG-HRP was found in multivesicular bodies and by 30 min appeared in basal vesicles, the frequency of the latter event increasing with time. No routing of IgG-HRP into Golgi regions or lysosomes could be detected. by 60 min IgG-HRP was found in a few caveolae of fetal endothelium of both terminal and intermediate villi. IgG-HRP was not found in intercellular clefts of the endothelium. The pattern of uptake and routing observed suggests a receptor-mediated transcytosis of IgG-HRP across the syncytiotrophoblast and a transcellular pathway through the endothelium.